ABSTRACT Inflamed bone fracture poses a significant clinical problem. In the United States, approximately 1.6 million bone fractures encounter prolonged healing or non-union each year, among which, the major population bearing with these clinical complications are patients with inflammatory conditions, e.g, elder patients, smoking, diabetic or rheumatoid arthritis (RA) patients. In these patients, the fracture risk is increased due to the poor bone quality, highlighting the potential deleterious role of chronic systemic inflammation in fracture repair. The overarching hypothesis of this proposal is that under inflammatory conditions, NF-?B, the principal mediator of inflammation, induces Rbpj? expression through downregulating Dnmt3b and its DNA methylation activity. We further hypothesize that Dnmt3b GOF or Rbpj? inhibition restores MPC differentiation and chondrocyte maturation that are reduced by inflammation during fracture repair. This hypothesis is supported by our preliminary data wherein we show that Dnmt3b is highly expressed in fracture callus during fracture repair and Dnmt3b is the major DNA methyltransferase (Dnmt) responsive to cytokine in MPCs and chondrocytes. Relevant to our proposal, we provide evidence that inflammatory signals inhibit Dnmt3b in MPCs and chondrocytes in an NF-?B-dependent manner. Consistently, mice with Dnmt3b loss-of-function (LOF) in MPCs and chondrocytes display delayed fracture repair; and Dnmt3b gain-of-function (GOF) in MPCs or chondrocytes shows protective effect from inflammation in vitro and accelerates fracture repair in mice. Mechanistically, MPC differentiation defect mediated by inflammation and Dnmt3b LOF coincide with upregulation of Rbpj? in MPCs and Rbpj? inhibition can restore differentiation capacity in vitro. In vitro mechanistic studies and in vivo LOF and GOF approaches will be used to modulate IKK2, Dnmt3b and Rbpj? expression in MPCs and chondrocytes to dissect its effects during fracture repair process. Three main Specific Aims are proposed. Specific Aim 1 will delineate the effect of constitutively active NF-?B signaling (IKK2ca), as the principal molecular driver of inflammation, on Dnmt3b expression and fracture repair. Specific Aim 2 will establish the effect of Dnmt3b GOF in MPCs and chondrocytes on accelerating fracture repair. Specific Aim 3 will delineate the mechanism by which Dnmt3b regulates downstream target, Rbpj?, during fracture repair. This work will enhance our understanding of mechanisms by which systemic inflammation (via the NF-?B pathway) affects the fracture healing process through Dnmt3b and identify downstream targets of Dnmt3b (such as Rbpj?) as novel candidates for therapeutic intervention.